Mycobacterium tuberculosis (Mtb) is the etiologic agent of the disease tuberculosis, which kills approximately 1.4 million people each year. Mtb's arduous treatment regimen and the recent emergence of increasingly drug-resistant Mtb strains have given rise to an urgent need for the development of new anti- tubercular drugs. One promising therapeutic target is the mycobacterial proteasome, a large proteolytic complex that is essential for both Mtb virulence and resistance to the host's antimicrobial nitric oxide response. Recent work has shown that proteins can be targeted for proteasomal degradation in Mtb by the pupylation pathway, a process that is functionally analogous to eukaryotic ubiquitination. Pupylation centers around the small protein modifier Pup (prokaryotic ubiquitin-like protein), which is covalently ligated to a protein to target it for proteolysis. Despite advances in our understanding of the Pup-proteasome system, no group has yet recapitulated robust degradation of a native substrate by the Mtb proteasome in vitro, implying that additional unknown factors are necessary for proteasomal activity. To search for such factors, we used a catalytically inactive proteasome trap that stabilizes proteasomal interactions to identify novel proteasome-associated proteins. A protein we are now referring to as PafE (proteasome accessory factor E) copurified with the proteasome trap in high abundance and co-occurs evolutionarily with the prokaryotic 20S core particle. Preliminary studies demonstrated that PafE resembles known proteasomal cofactors, as it forms multimeric rings and interacts with the 20S through a conserved carboxy-terminal motif. Thus, in an effort to enhance our understanding of the prokaryotic proteasome complex, the proposed research seeks to determine PafE's role in proteasome function through two specific aims. First, we will determine if PafE plays a role in proteasomal degradation in vivo. Through immunoblotting we will determine if PafE is required for the degradation of known pupylated proteasome substrates. In addition, we will utilize the proteasome trap to search for non-pupylated PafE-dependent substrates that copurify with the proteasome only in the presence of PafE. We will then determine if PafE is required for Mtb virulence in a mouse infection model. Second, we will determine if PafE has a direct role in proteasomal degradation in vitro. We will use recombinant 20S and PafE purified from E. coli to validate that binding occurs independent of any other factors. We will then perform in vitro reactions using purified components to test if PafE influences the proteasomal degradation rate of either a peptide substrate or the native Mtb proteasome substrate Pup~FabD. These studies will substantially enhance our understanding of the prokaryotic proteasome and the mechanisms that regulate its function. Because the Mtb proteasome is essential for normal growth and virulence, this work has strong implications for the elucidation of new therapeutic avenues to treat tuberculosis.